高雄激素促进人胚胎干细胞向男性原始生殖细胞分化的研究

High-level androgen enhances the differentiation of hESCs into male primordial germ cells

目的:多囊卵巢综合征(PCOS)母亲孕早期的高雄激素可能影响其男性子代成年后的生育力。本研究利用人胚胎干细胞(hESCs)体外分化的实验模型,观察不同浓度睾酮对hESCs向早期雄性生殖细胞分化的影响,探讨孕早期高雄激素暴露对男性子代原始生殖细胞储备及成年后生育力的潜在影响。方法:2μmol/L视黄酸(RA)体外诱导hESCs(46,XY)向雄性生殖细胞分化,同时添加0 mol/L、3×10^-7 mol/L、5×10^-7 mol/L、15×10^-7 mol/L、45×10^-7 mol/L、135×10^-7 mol/L睾酮,在分化的第0、4、7、14天收集细胞样品,分析不同分化阶段雄性生殖细胞特异基因的表达,比较分化进程、分化效率。结果:不同浓度睾酮处理的hESCs,同一分化阶段细胞形态无差异。原始生殖细胞的标志基因DAZL的表达峰值出现在分化的第4天,15×10^-7 mol/L、45×10^-7 mol/L、135×10^-7 mol/L睾酮处理组的DAZL的表达量与3×10^-7 mol/L睾酮处理组对比明显增加(P﹤0.01)。减数分裂期早期生殖细胞特异基因SCP3的表达在分化的第4天开始上调,45×10^-7 mol/L睾酮处理组与3×10^-7 mol/L、5×10^-7 mol/L睾酮处理组比较,SCP3的表达均明显上调(P﹤0.01);SCP3的表达在分化的第7天达到峰值,15×10^-7 mol/L、45×10^-7 mol/L、135×10^-7 mol/L睾酮处理组与3×10^-7 mol/L睾酮处理组相比,SCP3明显高表达(P﹤0.01)。免疫荧光、流式细胞分析结果显示,各组间分化第4天DAZL、分化第7天SCP3及VASA的表达量随着睾酮浓度升高而有升高趋势。分化第4天,雄激素受体(AR)的表达开始升高,并且可维持至分化第14天;在相同分化阶段,睾酮较高浓度组hESCs的AR表达量高于睾酮较低浓度组(但P>0.05)。结论:hESCs向早期雄性生殖细胞分化过程中高雄激素处理,诱导雄激素受体的提前表达,使hESCs向早期雄性生殖细胞分化提前,可能导致PCOS患者男性子代雄性原始生殖细胞储备不足,进而影响其成年后的生育力。hESCs可作为体外模型研究PCOS患者宫内高雄激素暴露对其男性子代生育力影响研究的新工具,并有益于男性不育症病因机制的研究。

Objective:To observe the effects of different concentrations of testosterone on the differentiation of human embryonic stem cells(hESCs)into early male germ cells and investigate the potential impact of high-level androgen exposure in early pregnancy in women with polycystic ovary syndrome(PCOS)on the fertility and primordial germ cell reserve of the male offspring in adulthood.Methods:We used 2μmol/L retinoic acid to induce the differentiation of hESCs(46,XY)into male germ cells in vitro and meanwhile treated them with testosterone(T)at 0 mol/L,3×10^-7 mol/L,5×10^-7 mol/L,15×10^-7 mol/L,45×10^-7 mol/L,and 135×10^-7 mol/L,respectively.We collected the cell samples at 0,4,7 and 14 days to determine the expressions of the specific genes and compare the differentiation process and efficiency of the male germ cells in different stages.Results:There was no difference in the morphology of the hESCs treated with different concentrations of testosterone in the same differentiation stage.The expression of the marker gene DAZL in the primordial germ cells peaked on the 4 th day of differentiation,significantly higher in the 15×10^-7,45×10^-7 and 135×10^-7 mol/L groups than in the 3×10^-7 mol/L group(P<0.05),and that of the specific gene SCP3 in the early-meiosis germ cells began to increase on the same day,more significantly in the 45×10^-7mol/L than in the 3×10^-7 mol/L and 5×10^-7 mol/L groups(P<0.01),and peaked on the 7 th day,dramatically higher in the 15×10^-7,45×10^-7 and 135×10^-7 mol/L groups than in the 3×10^-7 mol/L group(P<0.01).Immunofluorescence staining and flow cytometry showed a T concentration-dependent increase in the expression of DAZL at 4 days and those of SCP3 and VASA at 7 days.Moreover,the expression of the androgen receptor(AR)in the hESCs began to rise on the 4 th day and kept going up till the 14 th day,higher in the high-concentration than in the low-concentration T groups in the same stage of differentiation,though with no statistically significant difference(P>0.05).Conclusion:Exposure to high-level androgen during the differentiation of hESCs into early male germ cells can induce earlier expression of AR and earlier differentiation of hESCs into early male germ cells,which may result in insufficient reserve of male primary germ cells in the male offspring of PCOS women and affect their fertility after adulthood.hESCs can be used as an in vitro model to study the effects of intrauterine hyperandrogen on the reproductive development of male offspring in PCOS patients,which is also contributive to researches on the etiology of male infertility.